Honokiol analogs and their use in treating cancers

ABSTRACT

Compounds, pharmaceutical compositions including the compounds, and methods of preparation and use thereof are disclosed. The compounds are honokiol analogs. The compounds and compositions can be used to treat and/or prevent a wide variety of cancers, including drug resistant cancers. Representative honokiol analogs include diepoxide honokiol analogues. The compounds are believed to function, at least, by inhibiting angiogenesis and/or inducing apoptosis. Thus, the compounds are novel therapeutic agents for a variety of cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase under the provisions of 35U.S.C. §371 of International Application No. PCT/US08/61899 filed Apr.29, 2008, which in turn claims priority of U.S. Patent Application No.60/927,338 filed May 3, 2007 and U.S. Patent Application No. 60/934,322filed Jun. 13, 2007. The disclosures of such international applicationand U.S. priority applications are hereby incorporated herein byreference in their respective entireties, for all purposes.

This invention was made with governmental support under Grant No.AR47901, awarded by National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel methods and compositions for thetreatment of primary and metastatic cancers. These methods andcompositions use honokiol analogues. These compounds, and pharmaceuticalcompositions including the compounds, are particularly useful fortreating primary and metastatic cancers in humans. The invention alsoencompasses the varying modes of administration of the therapeuticcompounds or compositions.

BACKGROUND OF THE INVENTION

Natural products are a major source of small molecular weightangiogenesis inhibitors, and the transformed endothelial cell line SVRhas been used to screen natural product extracts to isolateanti-angiogenesis and anti-tumor compounds (Arbiser et al., J. Biol.Chem., Vol. 278, Issue 37, 35501-35507, Sep. 12, 2003).

Aqueous extracts of Magnolia grandiflora exhibit potent activity inthese SVR proliferation assays, and the small molecular weight compoundhonokiol is the active principle of magnolia extract. Honokiol has thefollowing formula:

Honokiol exhibits potent anti-proliferative activity against SVR cellsin vitro. In addition, honokiol demonstrates preferential inhibition ofprimary human endothelial cells compared with fibroblasts, and thisinhibition was antagonized by antibodies against TNFα-relatedapoptosis-inducing ligand. In vivo, honokiol is highly effective againstangiosarcoma in nude mice. Preclinical data suggests that honokiol is asystemically available and non-toxic inhibitor of angiogenesis, and alsopromotes apoptosis.

There remains a need for treatment of cancer that does not have theadverse effects generally caused by non-selectivity, of conventionalchemotherapeutic agents. While honokiol is an active compound, it wouldbe advantageous to develop honokiol analogues that are even more active.The present invention provides such analogues, as well as pharmaceuticalcompositions including the analogues, and methods of treating cancerusing the analogues.

SUMMARY OF THE INVENTION

Compounds, pharmaceutical compositions including the compounds, andmethods of preparation and use thereof are disclosed.

In one embodiment, the compounds are honokiol analogs, which can beformed by reacting one or both of the double bonds in honokiol withappropriate reactants to form cyclopropane, epoxide, thiirane, oraziridine rings. A core structure similar to honokiol, where one or bothof the benzene rings are replaced with a heteroaryl ring, can also beused. The linker between the aryl/heteroaryl rings and the cyclopropane,epoxide, thiirane, or aziridine rings is modified from that of honokiol,in that it can be extended from one to three carbons in length, and oneof the carbons replaced with an O, S, or amine. Additionally, one orboth of the hydroxyl groups on the central aryl/heteroaryl rings can beconverted to an alkyl phosphate ester, or a dichloroacetate ester.Representative compounds include honokiol diepoxide and honokiolmonoepoxide.

The synthesis, characterization and an evaluation of the anti-tumorpotential of these honokiol analogues is also disclosed. The honokiolanalogues inhibit angiogenesis and/or induce apoptosis, and thus areeffective at killing growing cancer cells.

Treatment with one or more of these compounds selectively kills cancercells, without killing healthy cells, thus providing a selectiveanti-cancer therapy. Most importantly, these compounds are potentagainst cancer cells that have become metastacized.

The pharmaceutical compositions include an effective amount of thecompounds described herein, along with a pharmaceutically acceptablecarrier or excipient. When employed in effective amounts, the compoundscan act as a therapeutic agent to prevent and/or treat a wide variety ofcancers, particularly metasticized cancers, and are believed to be bothsafe and effective in this role. Representative cancers that can betreated and/or prevented include melanoma, leukemia, non-small celllung, colon, central nervous system (CNS), renal, ovarian, breast andprostate cancer.

The foregoing and other aspects of the present invention are explainedin detail in the detailed description and examples set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the effect of honokiol diepoxide against tumorcells in vivo (average tumor volume).

DETAILED DESCRIPTION OF THE INVENTION

Compounds, pharmaceutical compositions including the compounds, andmethods of preparation and use thereof are disclosed.

The following definitions will be useful in understanding the metes andbounds of the invention as described herein.

As used herein, “alkyl” refers to straight chain or branched alkylradicals including C₁-C₈, preferably C₁-C₅, such as methyl, ethyl, orisopropyl; “substituted alkyl” refers to alkyl radicals further bearingone or more substituent groups such as hydroxy, alkoxy, aryloxy,mercapto, aryl, heterocyclo, halo, amino, carboxyl, carbamyl, cyano, andthe like; “alkenyl” refers to straight chain or branched hydrocarbonradicals including C₁-C₈, preferably C₁-C₅ and having at least onecarbon-carbon double bond; “substituted alkenyl” refers to alkenylradicals further bearing one or more substituent groups as definedabove; “cycloalkyl” refers to saturated or unsaturated, non-aromatic,cyclic ring-containing radicals containing three to eight carbon atoms,preferably three to six carbon atoms; “substituted cycloalkyl” refers tocycloalkyl radicals further bearing one or more substituent groups asdefined above; “aryl” refers to aromatic radicals having six to tencarbon atoms; “substituted aryl” refers to aryl radicals further bearingone or more substituent groups as defined above; “alkylaryl” refers toalkyl-substituted aryl radicals; “substituted alkylaryl” refers toalkylaryl radicals further bearing one or more substituent groups asdefined above; “arylalkyl” refers to aryl-substituted alkyl radicals;“substituted arylalkyl” refers to arylalkyl radicals further bearing oneor more substituent groups as defined above; “heterocyclyl” refers tosaturated or unsaturated cyclic radicals containing one or moreheteroatoms (e.g., O, N, S) as part of the ring structure and having twoto seven carbon atoms in the ring; “substituted heterocyclyl” refers toheterocyclyl radicals further bearing one or more substituent groups asdefined above.

I. Compounds

The compounds are honokiol analogs, prodrugs or metabolites of thesecompounds, and pharmaceutically acceptable salts thereof, wherein one orboth of the double bonds in honokiol has been replaced with acyclopropane, epoxide, thiirane, or aziridine moiety, optionally alongwith other structural modifications and optional substitutions.

In one embodiment, the compounds have the following formula:

wherein:

W is (CHR²)n, O—(CHR²)n, S—(CHR²)n, or NR²—(CHR²)n, optionallysubstituted with halogens, such as fluorine (i.e., (CF₂)n and the like),

X is O, S, or NR²,

Y is N or C bonded to a substituent, G,

R¹ is H, alkyl phosphate, dichloroacetate, trifluoromethyl, orvalproate,

R² is H, alkyl, aryl, arylalkyl, or alkylaryl, and when bonded tocarbon, halo, such as fluoro,

n is an integer from 1-4,

Representative substituents, G, include C₁₋₆ alkyl (includingcycloalkyl), alkenyl, heterocyclyl, aryl, heteroaryl, halo (e.g., F, Cl,Br, or I), —OR, —NR³R⁴, —CF₃, —CN, —NO₂, —C₂R³, —SR³, —N₃, —C(═O)NR³R⁴,—NR³C(═O)R³, —C(═O)R³, —C(═O)OR³, —OC(═O)R³, —OC(═O)NR³R⁴, —NR³C(═O)OR³,—SO₂R³, —SO₂NR³R⁴, and —NR³SO₂R³, where R³ and R⁴ are individuallyhydrogen, C₁₋₆ alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl,(such as benzyl);

z is an integer of from 0-3, but in any case cannot exceed the number ofcarbon atoms in the ring,

one or both of the aryl rings can be replaced with thiophene, pyrrole,or furan, and

one of the three membered rings can be replaced with a double bond(i.e., X represents a bond between the two carbons to which it isattached).

In one embodiment, a fluoro moiety is present ortho to one or both OR¹moieties. In one aspect of this embodiment, each Y is CF.

In another embodiment, the compounds have the following formula:

wherein W, X, R¹, R², G n and z are as defined above, and wherein one ofthe three membered rings can be replaced with a double bond.

In still another embodiment, the compounds have the following formula:

Wherein W, R1 and R2 are as defined above, and wherein one of theepoxide rings can be replaced with a double bond.

Representative individual compounds include the following:

The compounds shown above have a double bond that can be present ineither cis or trans (E or Z) form, and also a chiral carbon on theepoxide ring, which can be in either the R or S configuration, ormixtures thereof. The individual compounds representing the variousstereoisomeric forms and isomeric forms of these compounds are withinthe scope of the invention.

The compounds of any of the above formulas can be present in the form ofracemic mixtures or pure enantiomers, or occur in varying degrees ofenantiomeric excess, and racemic mixtures can be purified using knownchiral separation techniques.

The compounds can be in a free base form or in a salt form (e.g., aspharmaceutically acceptable salts). Examples of suitablepharmaceutically acceptable salts include inorganic acid addition saltssuch as sulfate, phosphate, and nitrate; organic acid addition saltssuch as acetate, galactarate, propionate, succinate, lactate, glycolate,malate, tartrate, citrate, maleate, fumarate, methanesulfonate,p-toluenesulfonate, and ascorbate; salts with an acidic amino acid suchas aspartate and glutamate; alkali metal salts such as sodium andpotassium; alkaline earth metal salts such as magnesium and calcium;ammonium salt; organic basic salts such as trimethylamine,triethylamine, pyridine, picoline, dicyclohexylamine, andN,N′-dibenzylethylenediamine; and salts with a basic amino acid such aslysine and arginine. The salts can be in some cases hydrates or ethanolsolvates. The stoichiometry of the salt will vary with the nature of thecomponents.

II. Methods of Preparing the Compounds

The compounds of Formula I have a bi-aryl, aryl-heteroaryl, orbi-heteroaryl core structure, where each ring includes a hydroxyl groupand a side chain. Depending on the definition of W, the side chain caninclude an ether, thioether, or amine bridge between the aryl/heteroarylring and the three membered ring. Depending on the definition of X, thethree membered ring can be a cyclopropane, epoxide, thiirane, oraziridine.

Generally, methods for producing bi-aryl, aryl-heteroaryl, orbi-heteroaryl core structures are known, and involve coupling chemistrybetween an aryl halide and an aryl organometallic compound, such as anaryl lithium or aryl zinc halide. Hydroxy groups present in appropriatepositions on the aryl/heteroaryl rings can be protected during thecoupling chemistry using known protecting groups.

Ideally, the aryl/heteroaryl rings either include an appropriate sidechain or side chain precursor, or include appropriate functionality toattach the side chain. A double bond can serve as a precursor for acyclopropane, epoxide, thiirane, or aziridine ring, as discussed in moredetail below.

Accordingly, using known starting materials, one can couple twoappropriately substituted aryl rings, an aryl ring and a heteroarylring, or two heteroaryl rings, attach a side chain or side chainprecursor, if the side chain or side chain precursor is not alreadyattached, and convert one or more side chain precursors (i.e., doublebonds) into cyclopropane, epoxide, thiirane, or aziridine rings.Representative processes for effecting these conversions are describedin detail below.

Synthesis of Honokiol Analogues Using Honokiol as a Starting Material

Honokiol can be used as a starting material for certain honokiolanalogues, principally those in which the modification to honokiol isthe conversion of one or both of the double bonds to a cyclopropane,epoxide, thiirane, or aziridine ring. The chemistry for converting thedouble bonds to these rings is discussed in more detail below.

Synthesis of Bi-Phenyls, Phenyl-Heteroaryl, and Bi-Heteroaryl Rings

Biaryl, aryl-heteroaryl, and bi-heteroaryl rings can be prepared, forexample, using the Negishi coupling reaction. This reaction involves thenickel- or palladium-catalyzed coupling of organozinc compounds withvarious halides (aryl and heteroaryl halides). For example,2-bromo-pyridine can be reacted with t-butyl lithium to produce thelithio salt, and this lithio salt reacted with zinc chloride to producethe 2-zinc chloride salt. This zinc chloride salt can be coupled, forexample, with 2-chloropyridine to produce a bi-pyridine (i.e., a2,2′-bipyridine).

The Negishi coupling reaction involves formation of organometallicsalts, and any hydroxyl groups present in the aryl/heteroaryl ring willbe deprotonated unless they are protected with suitable protectinggroups. Further, any dichloroacetate and alkyl phosphate ester analoguesof these hydroxyl groups will likely be reacted with the organometallicsalts, so it is best to prepare any dichloroacetate and alkyl phosphateester analogues after the Negishi coupling step.

Biphenyl rings can typically be prepared by reacting fluoroaryl ringswith an aryl lithium compound. The reaction is believed to proceed by abenzyne intermediate.

Compounds where W is C₁₋₄ Alkyl or Haloalkyl

Compounds where W is C₁₋₄ alkyl or haloalkyl, such as perfluoroalkyl orany fluorinated analogue with from one fluorine to perfluorination, canbe prepared via a number of methods, including either a) having anappropriate C₁₋₄ alkyl moiety, with a double bond attached at theterminal end of the side chain, present during the coupling of thearyl/heteroaryl rings to form the biaryl, aryl-heteroaryl, orbi-heteroaryl rings, or b) including a functional group on thearyl/heteroaryl rings that can be converted to a C₁₋₄ alkyl moiety, witha double bond attached at the terminal end of the side chain.

For example, if a halide is present on one of the aryl/heteroaryl rings,it can be reacted, for example, using conventional coupling chemistry,with a halo-alkene (such as an allyl halide (also known as a3-halo-prop-1-ene, for example, allyl bromide), 4-halo-but-1-ene,5-halo-pent-1-ene, or 6-halo-hex-1-ene), to form an aryl-alkene orheteroaryl-alkene intermediate. Following the coupling reaction, thedouble bond can be converted to a suitable three membered ring, such asa cyclopropane, epoxide, thiirane, or aziridine. The doublebond-containing moieties listed above would produce a three memberedring with a (CH₂) moiety, but if other functionalization is desired, oneof the hydrogens on the ═CH₂ terminus of the halo-alkene could bereplaced with an alkyl, aryl, alkylaryl, or arylalkyl group. Such doublebond-containing materials are either well known to those of skill in theart, or can be easily prepared using conventional chemistry.

If one or more double bond-containing moieties are present during thecoupling of the aryl/heteroaryl rings, they will not interfere with norbe destroyed during the coupling chemistry. Similarly, if a cyclopropanering-containing side chain is present on the aryl/heteroaryl ring duringthe coupling chemistry, the ring will not be adversely affected duringthe coupling chemistry. However, if a side chain when X O, S, or NR²were present during the coupling chemistry, these groups might beadversely affected, so it is preferred to first provide the double bondmoiety, and then to convert it to the desired cyclopropane, thiirane, oraziridine moiety.

Compounds where W is O—(CHR²)n, S—(CHR²)n, or NR²—(CHR²)n

Compounds where W is O—(CHR²)n, S—(CHR²)n, or NR²—(CHR²)n can beprepared via a number of methods, including either a) having anappropriate O—(CHR²)n, S—(CHR²)n, or NR²—(CHR²)n alkyl moiety, with adouble bond attached at the terminal end of the side chain, presentduring the coupling of the aryl/heteroaryl rings to form the biaryl,aryl-heteroaryl, or bi-heteroaryl rings, or b) including a protectedhydroxyl, thiol, or amine group on the aryl/heteroaryl rings that can bedeprotected and subsequently converted to an O—(CHR²)n, S—(CHR²)n, orNR²—(CHR²)n moiety, with a double bond attached at the terminal end ofthe side chain. The latter can be accomplished, for example, by reactionof a hydroxyl, thiol, or amine with an alkenyl bromide (as above) toform an ether, thioether, or amine linkage.

In some embodiments, it is desired to incorporate halogens, such asflourines, into the alkyl group (i.e., (CF₂)n). Such fluorinatedmoieties can readily be incorporated, using techniques known to those ofskill in the art.

Conversion of Double Bonds to Cyclopropane Rings

Following the attachment of double bond-containing side chains, one orboth of the double bonds can be converted to cyclopropane rings. Thecyclopropane rings can include a CH₂ moiety, or can be substituted withone or two methyl groups.

The derivatives described herein include derivatives in which one orboth of the double bonds is replaced with a (unsubstituted, monoalkyl ordialkyl, where alkyl can be substituted or unsubstituted, and ispreferably methyl) cyclopropyl group.

The synthesis of alkyl, such as methyl, dialkyl, such as dimethyl andunsubstituted cyclopropane derivatives is well known to those of skillin the art, and involves, for example, bromoform reaction to form thedibromocyclopropane derivative, followed by stoichiometric reaction witha hydride or an alkyl-lithium. An aryl-lithium will provide arylsubstitution on the cyclopropane ring. If fluoro-substitution is desiredon the cyclopropane ring, it can be provided, for example, by displacingthe bromines with fluorines using known chemistry.

Conversion of Double Bonds to Epoxide Rings

Following the attachment of double bond-containing side chains, one orboth of the double bonds can be converted to epoxide rings. The epoxiderings can be formed, for example, by reaction of the double bond withm-chloroperbenzoic acid. Alternatively, the epoxide rings can be formedby halohydrogenation of the double bond to form halohydrins, followed bythe addition of base. Halohydrins are typically prepared by addingaqueous hypochlorous acid (HOCl) or hypobromous acid (HOBr) to alkenes,often by using aqueous solutions of the halogen, where the reactionproceeds by formation of the intermediate halonium ion. The basedeprotonates the hydroxyl group, which then nucleophilically displacesthe halide to form the epoxide ring. The choice of reaction conditionscan be made depending on the susceptibility of the other substituents onthe intermediate to such reaction conditions.

Conversion of Double Bonds to Thiirane Rings

Following the attachment of double bond-containing side chains, one orboth of the double bonds can be converted to thiirane rings. Thethiirane rings can be formed, for example, by bromination of the doublebond, followed by S′-substitution in sodium sulfides (see for example,Choi J et al (1995) Bull. Korean. Chem. Soc., 16, 189-190, ConvenientSynthesis of Symmetrical Sulfides from Alkyl Halides and Epoxides).

Conversion of Double Bonds to Aziridine Rings

Following the attachment of double bond-containing side chains, one orboth of the double bonds can be converted to aziridine rings. Theaziridine rings can be formed, for example, by selective aziridinationof olefins with p-toluenesulfonamide catalyzed by dirhodium(II)caprolactamate. Aziridine formation occurs through aminobromination andsubsequent base-induced ring closure. See, for example, A. J. Catino, J.M. Nichols, R. E. Forslund, M. P. Doyle, Org. Lett., 2005, 7, 2787-2790.

Protection and Deprotection of Hydroxy Groups

As discussed herein, hydroxyl groups present on the aryl/heteroarylrings may need to be protected during portions of the synthesis, anddeprotected at a later time. Protecting groups, and methods for theirremoval, are well known to those of skill in the art, and are describedfor example, in T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 3^(rd) Edition, John Wiley & Sons, New York (1999).

Conversion of Hydroxy Groups to Phosphate Esters

One or both of the hydroxyl groups present on the aryl/heteroaryl rings,on honokiol itself or on a honokiol analogue, can be converted tophosphate esters (i.e., where R1 is a phosphate ester moiety), includingalkyl phosphates, by reacting the hydroxyl group(s) with aphosphorylating agent, such as phosphoric anhydride, polyphosphoricacid, phosphorous oxychloride, and phosphorous pentoxide.

Conversion of Hydroxy Groups to Dichloroacetate or Valproic Acid Esters

One or both of the hydroxyl groups present on the aryl/heteroaryl rings,on honokiol itself or on a honokiol analogue, can be converted todichloroacetate and/or valproic acid esters (i.e., where R1 is adichloroacetate moiety, Cl₂CHC(O)— or a valproate moiety), by reactingthe hydroxyl group(s) with dichloroacetic acid, valproic acid, anactivated forms thereof. The dichloroacetic acid or valproic acid can becoupled to a hydroxyl group directly, with an acid catalyst andsubsequent formation of water (typically removed by azeotropicdistillation), or by reaction with an acid halide or anhydride form ofdichloroacetic acid or valproic acid, typically in the presence of atertiary amine such as triethylamine.

Conversion of Hydroxy Groups to Trifluoromethyl Ethers

Starting with honokiol or the analogues thereof, one can readily convertone or both of the hydroxyl groups to a trifluoromethyl ether, usingtechniques known to those of skill in the art. For example, an alkoxidesalt of honokiol or an analogue thereof can be reacted withtrifluoroiodomethane to form the trifluoromethyl ether.

The starting materials used to make the honokiol analogues describedherein are either commercially available, or can be prepared fromcommercially available starting materials. Those that are notcommercially available can be made by a variety of syntheticmethodologies, related to the particular moieties and the particularsubstitution desired. The variation in synthetic methodology will bereadily apparent to those of skill in the art of organic synthesis.

Those skilled in the art will readily understand that incorporation ofother substituents onto the aromatic or heteroaromatic rings used as astarting material to prepare the honokiol analogues, and other positionsin the honokiol framework, can be readily realized. Such substituentscan provide useful properties in and of themselves or serve as a handlefor further synthetic elaboration.

Substituents typically can be added to a phenyl ring, heteroaryl ring,bi-phenyl moiety, phenyl-heteroaryl and/or bi-heteroaryl moiety beforeadding the side chains.

A number of other analogs, bearing substituents in the diazotizedposition of the aryl/heteroaryl rings, can be synthesized from thecorresponding amino compounds, via diazonium salt intermediates. Thediazonium salt intermediates can be prepared using known chemistry, forexample, as described above.

Nitration of an aryl or heteroaryl results, followed by reaction with anitrite salt, typically in the presence of an acid, produces an aminefunctionality on the aryl/heteroaryl ring. Other substituted analogs canbe produced from diazonium salt intermediates, including, but are notlimited to, hydroxy, alkoxy, fluoro, chloro, iodo, cyano, and mercapto,using general techniques known to those of skill in the art. Forexample, hydroxy-aryl/heteroaryl analogues can be prepared by reactingthe diazonium salt intermediate with water. Likewise, alkoxy honokiolanalogues can be made by reacting the diazonium salt with alcohols. Thediazonium salt intermediates can also be used to synthesize cyano orhalo compounds, as will be known to those skilled in the art. Mercaptosubstitutions can be obtained using techniques described in Hoffman etal., J. Med. Chem. 36: 953 (1993). The mercaptan so generated can, inturn, be converted to an alkylthio substitutuent by reaction with sodiumhydride and an appropriate alkyl bromide. Subsequent oxidation wouldthen provide a sulfone. Acylamido analogs of the aforementionedcompounds can be prepared by reacting the corresponding amino compoundswith an appropriate acid anhydride or acid chloride using techniquesknown to those skilled in the art of organic synthesis.

Hydroxy-substituted analogs can be used to prepare correspondingalkanoyloxy-substituted compounds by reaction with the appropriate acid,acid chloride, or acid anhydride. Likewise, the hydroxy compounds areprecursors of both the aryloxy and heteroaryloxy via nucleophilicaromatic substitution at electron deficient aromatic rings. Suchchemistry is well known to those skilled in the art of organicsynthesis. Ether derivatives can also be prepared from the hydroxycompounds by alkylation with alkyl halides and a suitable base or viaMitsunobu chemistry, in which a trialkyl- or triarylphosphine anddiethyl azodicarboxylate are typically used. See Hughes, Org. React.(N.Y.) 42: 335 (1992) and Hughes, Org. Prep. Proced. Int. 28: 127 (1996)for typical Mitsunobu conditions.

Cyano-substituted analogs can be hydrolyzed to afford the correspondingcarboxamido-substituted compounds. Further hydrolysis results information of the corresponding carboxylic acid-substituted analogs.Reduction of the cyano-substituted analogs with lithium aluminum hydrideyields the corresponding aminomethyl analogs. Acyl-substituted analogscan be prepared from corresponding carboxylic acid-substituted analogsby reaction with an appropriate alkyllithium using techniques known tothose skilled in the art of organic synthesis.

Carboxylic acid-substituted analogs can be converted to thecorresponding esters by reaction with an appropriate alcohol and acidcatalyst. Compounds with an ester group can be reduced with sodiumborohydride or lithium aluminum hydride to produce the correspondinghydroxymethyl-substituted analogs. These analogs in turn can beconverted to compounds bearing an ether moiety by reaction with sodiumhydride and an appropriate alkyl halide, using conventional techniques.Alternatively, the hydroxymethyl-substituted analogs can be reacted withtosyl chloride to provide the corresponding tosyloxymethyl analogs,which can be converted to the corresponding alkylaminoacyl analogs bysequential treatment with thionyl chloride and an appropriate alkylamineCertain of these amides are known to readily undergo nucleophilic acylsubstitution to produce ketones.

Hydroxy-substituted analogs can be used to prepare N-alkyl- orN-arylcarbamoyloxy-substituted compounds by reaction with N-alkyl- orN-arylisocyanates. Amino-substituted analogs can be used to preparealkoxycarboxamido-substituted compounds and urea derivatives by reactionwith alkyl chloroformate esters and N-alkyl- or N-arylisocyanates,respectively, using techniques known to those skilled in the art oforganic synthesis.

Similarly, benzene rings (and pyridine, pyrimidine, pyrazine, and otherheteroaryl rings) can be substituted using known chemistry, includingthe reactions discussed above. For example, the nitro group onnitrobenzene can be reacted with sodium nitrite to form the diazoniumsalt, and the diazonium salt manipulated as discussed above to form thevarious substituents on a benzene ring.

III. Pharmaceutical Compositions

The compounds described herein can be incorporated into pharmaceuticalcompositions and used to prevent a condition or disorder in a subjectsusceptible to such a condition or disorder, and/or to treat a subjectsuffering from the condition or disorder. The pharmaceuticalcompositions described herein include one or more of the honokiolanalogues described herein, and/or pharmaceutically acceptable saltsthereof. Optically active compounds can be employed as racemic mixtures,as pure enantiomers, or as compounds of varying enantiomeric purity.

The manner in which the compounds are administered can vary. Thecompositions are preferably administered orally (e.g., in liquid formwithin a solvent such as an aqueous or non-aqueous liquid, or within asolid carrier). Preferred compositions for oral administration includepills, tablets, capsules, caplets, syrups, and solutions, including hardgelatin capsules and time-release capsules. Compositions may beformulated in unit dose form, or in multiple or subunit doses. Preferredcompositions are in liquid or semisolid form. Compositions including aliquid pharmaceutically inert carrier such as water or otherpharmaceutically compatible liquids or semisolids may be used. The useof such liquids and semisolids is well known to those of skill in theart.

The compositions can also be administered via injection, i.e.,intraveneously, intramuscularly, subcutaneously, intraperitoneally,intraarterially, intrathecally; and intracerebroventricularly.Intravenous administration is a preferred method of injection. Suitablecarriers for injection are well known to those of skill in the art, andinclude 5% dextrose solutions, saline, and phosphate buffered saline.The compounds can also be administered as an infusion or injection(e.g., as a suspension or as an emulsion in a pharmaceuticallyacceptable liquid or mixture of liquids).

The formulations may also be administered using other means, forexample, rectal administration. Formulations useful for rectaladministration, such as suppositories, are well known to those of skillin the art. The compounds can also be administered by inhalation (e.g.,in the form of an aerosol either nasally or using delivery articles ofthe type set forth in U.S. Pat. No. 4,922,901 to Brooks et al., thedisclosure of which is incorporated herein in its entirety); topically(e.g., in lotion form); or transdermally (e.g., using a transdermalpatch, using technology that is commercially available from Novartis andAlza Corporation). Although it is possible to administer the compoundsin the form of a bulk active chemical, it is preferred to present eachcompound in the form of a pharmaceutical composition or formulation forefficient and effective administration.

The compounds can be incorporated into drug delivery devices such asnanoparticles, microparticles, microcapsules, and the like.Representative microparticles/nanoparticles include those prepared withcyclodextrins, such as pegylated cyclodextrins, liposomes, includingsmall unilamellar vesicles, and liposomes of a size designed to lodge incapillary beds around growing tumors. Suitable drug delivery devices aredescribed, for example, in Heidel J D, et al., Administration innon-human primates of escalating intravenous doses of targetednanoparticles containing ribonucleotide reductase subunit M2 siRNA, ProcNatl Acad Sci USA. 2007 Apr. 3; 104(14):5715-21; Wongmekiat et al.,Preparation of drug nanoparticles by co-grinding with cyclodextrin:formation mechanism and factors affecting nanoparticle formation, ChemPharm Bull (Tokyo). 2007 March; 55(3):359-63; Bartlett and Davis,Physicochemical and biological characterization of targeted, nucleicacid-containing nanoparticles, Bioconjug Chem. 2007 March-April;18(2):456-68; Villalonga et al., Amperometric biosensor for xanthinewith supramolecular architecture, Chem Commun (Camb). 2007 Mar. 7;(9):942-4; Defaye et al., Pharmaceutical use of cyclodextrines:perspectives for drug targeting and control of membrane interactions,Ann Pharm Fr. 2007 January; 65(1):33-49; Wang et al., Synthesis ofOligo(ethylenediamino)-beta-Cyclodextrin Modified Gold Nanoparticle as aDNA Concentrator; Mol Pharm. 2007 March-April; 4(2):189-98; Xia et al.,Controlled synthesis of Y-junction polyaniline nanorods and nanotubesusing in situ self-assembly of magnetic nanoparticles, J NanosciNanotechnol., 2006 December; 6(12):3950-4; and Nijhuis et al.,Room-temperature single-electron tunneling in dendrimer-stabilized goldnanoparticles anchored at a molecular printboard, Small. 2006 December;2(12):1422-6.

Exemplary methods for administering such compounds will be apparent tothe skilled artisan. The usefulness of these formulations may depend onthe particular composition used and the particular subject receiving thetreatment. These formulations may contain a liquid carrier that may beoily, aqueous, emulsified or contain certain solvents suitable to themode of administration.

The compositions can be administered intermittently or at a gradual,continuous, constant or controlled rate to a warm-blooded animal (e.g.,a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey),but advantageously are administered to a human being. In addition, thetime of day and the number of times per day that the pharmaceuticalformulation is administered can vary.

Preferably, the compositions are administered such that activeingredients interact with regions where cancer cells are located. Thecompounds described herein are very potent at treating these cancers.

In certain circumstances, the compounds described herein can be employedas part of a pharmaceutical composition with other compounds intended toprevent or treat a particular cancer, i.e., combination therapy. Inaddition to effective amounts of the compounds described herein, thepharmaceutical compositions can also include various other components asadditives or adjuncts.

Combination Therapy

The combination therapy may be administered as (a) a singlepharmaceutical composition which comprises a honokiol analogue asdescribed herein, at least one additional pharmaceutical agent describedherein, and a pharmaceutically acceptable excipient, diluent, orcarrier; or (b) two separate pharmaceutical compositions comprising (i)a first composition comprising a honokiol analogue as described hereinand a pharmaceutically acceptable excipient, diluent, or carrier, and(ii) a second composition comprising at least one additionalpharmaceutical agent described herein and a pharmaceutically acceptableexcipient, diluent, or carrier. The pharmaceutical compositions can beadministered simultaneously or sequentially and in any order.

In use in treating or preventing cancer, the honokiol analoguesdescribed herein can be administered together with at least one otherchemotherapeutic agent as part of a unitary pharmaceutical composition.Alternatively, the honokiol analogues can be administered apart from theother anticancer chemotherapeutic agent. In this embodiment, thehonokiol analogues and the at least one other anticancerchemotherapeutic agent are administered substantially simultaneously,i.e. the compounds are administered at the same time or one after theother, so long as the compounds reach therapeutic levels for a period oftime in the blood.

Combination therapy involves administering a honokiol analogue, asdescribed herein, or a pharmaceutically acceptable salt or prodrug of acompound described herein, in combination with at least one anti-cancerchemotherapeutic agent, ideally one which functions by a differentmechanism (i.e., VEGF inhibitors, alkylating agents, and the like).

Examples of known anticancer agents which can be used for combinationtherapy include, but are not limited to alkylating agents, such asbusulfan, cis-platin, mitomycin C, and carboplatin; antimitotic agents,such as colchicine, vinblastine, paclitaxel, and docetaxel; topo Iinhibitors, such as camptothecin and topotecan; topo II inhibitors, suchas doxorubicin and etoposide; RNA/DNA antimetabolites, such as5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites,such as 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;and antibodies, such as Herceptin® and Rituxan®. Other known anti-canceragents, which can be used for combination therapy, include arsenictrioxide, gamcitabine, melphalan, chlorambucil, cyclophosamide,ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin,bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoicacid, tamoxifen and alanosine. Other classes of anti-cancer compoundsthat can be used in combination with the honokiol analogues aredescribed below.

The honokiol analogues can be combined with alpha-1-adrenoceptorantagonists, such as doxazosin, terazosin, and tamsulosin, which caninhibit the growth of prostate cancer cell via induction of apoptosis(Kyprianou, N., et al., Cancer Res 60:4550 4555, (2000)).

Sigma-2 receptors are expressed in high densities in a variety of tumorcell types (Vilner, B. J., et al., Cancer Res. 55: 408 413 (1995)) andsigma-2 receptor agonists, such as CB-64D, CB-184 and haloperidol,activate a novel apoptotic pathway and potentiate antineoplastic drugsin breast tumor cell lines. (Kyprianou, N., et al., Cancer Res. 62:313322 (2002)). Accordingly, the honokiol analogues can be combined with atleast one known sigma-2 receptor agonists, or a pharmaceuticallyacceptable salt of said agent.

The honokiol analogues can be combined with lovastatin, a HMG-CoAreductase inhibitor, and butyrate, an inducer of apoptosis in the Lewislung carcinoma model in mice, can potentiate antitumor effects(Giermasz, A., et al., Int. J. Cancer 97:746 750 (2002)). Examples ofknown HMG-CoA reductase inhibitors, which can be used for combinationtherapy include, but are not limited to, lovastatin, simvastatin,pravastatin, fluvastatin, atorvastatin and cerivastatin, andpharmaceutically acceptable salts thereof.

Certain HIV protease inhibitors, such as indinavir or saquinavir, havepotent anti-angiogenic activities and promote regression of Kaposisarcoma (Sgadari, C., et al., Nat. Med. 8:225 232 (2002)). Accordingly(in addition to forming honokiol analogues of these compounds), thehonokiol analogues can be combined with HIV protease inhibitors, or apharmaceutically acceptable salt of said agent. Representative HIVprotease inhibitors include, but are not limited to, amprenavir,abacavir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir,tipranavir, ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632.

Synthetic retinoids, such as fenretinide (N-(4-hydroxyphenyl)retinamide,4HPR), can have good activity in combination with other chemotherapeuticagents, such as cisplatin, etoposide or paclitaxel in small-cell lungcancer cell lines (Kalemkerian, G. P., et al., Cancer Chemother.Pharmacol. 43:145 150 (1999)). 4HPR also was reported to have goodactivity in combination with gamma-radiation on bladder cancer celllines (Zou, C., et al., Int. J. Oncol. 13:1037 1041 (1998)).Representative retinoids and synthetic retinoids include, but are notlimited to, bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, .alpha.-difluoromethylornithine, ILX23-7553, fenretinide, andN-4-carboxyphenyl retinamide.

Proteasome inhibitors, such as lactacystin, exert anti-tumor activity invivo and in tumor cells in vitro, including those resistant toconventional chemotherapeutic agents. By inhibiting NF-kappaBtranscriptional activity, proteasome inhibitors may also preventangiogenesis and metastasis in vivo and further increase the sensitivityof cancer cells to apoptosis (Almond, J. B., et al., Leukemia 16:433 443(2002)). Representative proteasome inhibitors include, but are notlimited to, lactacystin, MG-132, and PS-341.

Tyrosine kinase inhibitors, such as STI571 (Imatinib mesilate,Gleevec®), have potent synergetic effects in combination with otheranti-leukemic agents, such as etoposide (Liu, W. M., et al. Br. J.Cancer 86:1472 1478 (2002)). Representative tyrosine kinase inhibitorsinclude, but are not limited to, Gleevec®, ZD1839 (Iressa®), SH268,genistein, CEP2563, SU6668, SU11248, and EMD121974.

Prenyl-protein transferase inhibitors, such as farnesyl proteintransferase inhibitor R115777, possess antitumor activity against humanbreast cancer (Kelland, L. R., et. al., Clin. Cancer Res. 7:3544 3550(2001)). Synergy of the protein farnesyltransferase inhibitor SCH66336and cisplatin in human cancer cell lines also has been reported (Adjei,A. A., et al., Clin. Cancer. Res. 7:1438 1445 (2001)). Prenyl-proteintransferase inhibitors, including farnesyl protein transferaseinhibitor, inhibitors of geranylgeranyl-protein transferase type I(GGPTase-I) and geranylgeranyl-protein transferase type-II, or apharmaceutically acceptable salt of said agent, can be used incombination with the honokiol analogues described herein. Examples ofknown prenylprotein transferase inhibitors include, but are not limitedto, R115777, SCH66336, L-778,123, BAL9611 and TAN-1813.

Cyclin-dependent kinase (CDK) inhibitors, such as flavopiridol, havepotent, often synergetic, effects in combination with other anticanceragents, such as CPT-11, a DNA topoisomerase I inhibitor in human coloncancer cells (Motwani, M., et al., Clin. Cancer Res. 7:4209 4219,(2001)). Representative cyclin-dependent kinase inhibitors include, butare not limited to, flavopiridol, UCN-01, roscovitine and olomoucine.

Certain COX-2 inhibitors are known to block angiogenesis, suppress solidtumor metastases, and slow the growth of implanted gastrointestinalcancer cells (Blanke, C. D., Oncology (Hunting) 16(No. 4 Suppl. 3):17 21(2002)). Representative COX-2 inhibitors include, but are not limitedto, celecoxib, valecoxib, and rofecoxib.

Any of the above-mentioned compounds can be used in combination therapywith the honokiol analogues. Additionally, many of these compounds canbe converted to honokiol analogues by reaction of ketone, aldehyde,hydroxyl, thiol, and/or amine functional groups on the compounds usingthe chemistry described herein. The honokiol analogues of thesecompounds are within the scope of this invention.

Further, the honokiol analogues can be targeted to a tumor site byconjugation with therapeutically useful antibodies, such as Herceptin®or Rituxan®, growth factors, such as DGF, NGF; cytokines, such as IL-2,IL-4, or any molecule that binds to the cell surface. The antibodies andother molecules will deliver a compound described herein to its targetsand make it an effective anticancer agent. The bioconjugates can alsoenhance the anticancer effect of therapeutically useful antibodies, suchas Herceptin® or Rituxan®.

The compounds can also be used in conjunction with surgical tumorremoval, by administering the compounds before and/or after surgery, andin conjunction with radiation therapy, by administering the compoundsbefore, during, and/or after radiation therapy.

The appropriate dose of the compound is that amount effective to preventoccurrence of the symptoms of the disorder or to treat some symptoms ofthe disorder from which the patient suffers. By “effective amount”,“therapeutic amount” or “effective dose” is meant that amount sufficientto elicit the desired pharmacological or therapeutic effects, thusresulting in effective prevention or treatment of the disorder.

When treating cancers, an effective amount of the honokiol analogue isan amount sufficient to suppress the growth of the tumor(s), and,ideally, is a sufficient amount to shrink the tumor, and, more ideally,to destroy the tumor. Cancer can be prevented, either initially, or fromre-occurring, by administering the compounds described herein in aprophylactic manner. Preferably, the effective amount is sufficient toobtain the desired result, but insufficient to cause appreciable sideeffects.

The effective dose can vary, depending upon factors such as thecondition of the patient, the severity of the cancer, and the manner inwhich the pharmaceutical composition is administered. The effective doseof compounds will of course differ from patient to patient, but ingeneral includes amounts starting where desired therapeutic effectsoccur but below the amount where significant side effects are observed.

The compounds, when employed in effective amounts in accordance with themethod described herein, are selective to certain cancer cells, but donot significantly affect normal cells.

For human patients, the effective dose of typical compounds generallyrequires administering the compound in an amount of at least about 1,often at least about 10, and frequently at least about 25 μg/24hr/patient. The effective dose generally does not exceed about 500,often does not exceed about 400, and frequently does not exceed about300 μg/24 hr/patient. In addition, administration of the effective doseis such that the concentration of the compound within the plasma of thepatient normally does not exceed 500 ng/mL and frequently does notexceed 100 ng/mL.

IV. Methods of Using the Compounds and/or Pharmaceutical Compositions

The compounds described herein, and pharmaceutical compositionsincluding the compounds, can be used to treat cancers. Representativedisorders that can be treated include neoplasms, such as hemangiomas,and malignant tumors, for example, those which arise in the setting ofautocrine loops involving vascular endothelial growth factor (VEGF) andits major mitogenic receptor vascular endothelial growth factor receptor2. Representative malignant tumors include malignant endothelial tumorssuch as melanoma. Additional cancers that can be treated include, butnot limited to human sarcomas and carcinomas, e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease, and malignant forms of these cancers.

In some embodiments, the patient already has cancer and is undergoingtreatment for the cancer, and may or may not have tumor metastasis(i.e., secondary cancer).

The cancer may be manifested in the form of a tumor, such as a tumor ofepithelial tissue, lymphoid tissue, connective tissue, bone, or centralnervous system.

The compounds can also be used as adjunct therapy in combination withexisting therapies in the management of the aforementioned types ofcancers. In such situations, it is preferably to administer the activeingredients to in a manner that optimizes effects upon cancer cells,including drug resistant cancer cells, while minimizing effects uponnormal cell types. While this is primarily accomplished by virtue of thebehavior of the compounds themselves, this can also be accomplished bytargeted drug delivery and/or by adjusting the dosage such that adesired effect is obtained without meeting the threshold dosage requiredto achieve significant side effects.

Treatment of Osteoporosis

The honokiol analogues can also be used to treat osteoporosis. Thecytokine RANKL (receptor activator of NF-κB ligand) causes osteoporosisby activating osteoclasts. Honokiol is known to inhibit RANKL activityby potentiating apoptosis, suppresses osteoclastogenesis, and inhibitsinvasion through modulation of nuclear factor-kappaB activation pathway(see, for example, Mol Cancer Res. 2006 September; 4(9):621-33). Thehonokiol analogues treat osteoporosis via the same mechanism.

Treatment of Inflammatory Disorders

The honokiol analogues described herein are useful for treating orpreventing inflammatory disorders. Reactive oxygen drives NFkB ininflammatory disorders such as rheumatoid arthitis, asthma, psoriasis,excema, lupus, scleroderma, certain heart diseases such atherosclerosisand coronary artery disease, and the like. Because the honokiolanalogues are effective at inhibiting production of reactive oxygenspecies, they are active against inflammatory disorders.

The honokiol analogues also inhibit certain inflammatory signals, andcan alleviate inflammatory disorders such as inflammatory arthritis byinhibiting these signals. For example, CD40 and its Epstein Barr viralmimic, LMP1, have been implicated in exacerbation of chronic autoimmunedisease, and the honokiol analogues inhibit CD40 and LMP1 inflammatorysignaling mechanisms. The effectiveness of the analogues at treatinginflammatory disorders can be shown by their ability to stabilize theseverity of symptomatic collagen-induced arthritis in both CD40-LMP1transgenic mice and their congenic C57Bl/6 counterparts. Theanti-inflammatory effects of these compounds can also be measured, forexample, in mouse B cell lines expressing the hCD40-LMP1 chimericreceptor, by measuring CD40 and LMP1-mediated NFkB and AP-1 activation,and the concomitant decrease in TNF-α and IL-6. The anti-inflammatoryproperties of these honokiol analogues can be used to block theautoimmune response.

Rheumatoid arthritis (RA) is considered the most common systemicautoimmune disease, but other disorders, such as hypothyroidism,systemic lupus erythematosus (SLE), and the like can also be treatedusing the honokiol analogues. A number of conditions are associated withchronic inflammation and elevated levels of TNF-α and IL-6, includingrheumatoid arthritis, heart disease, and cancer. Numerousgastrointestinal disorders are caused by inflammation, including, butnot limited to, Chrohn's disease, irritable bowel syndrome, andinflammatory bowel syndrome, and these disorders can also be treatedand/or prevented using the compounds described herein.

There is a suggested link between rheumatoid arthritis and chronicinflammation due to the re-activation of Epstein-Barr virus (EBV), whichlatently infects a proportion of memory B cells in >90% of the world'spopulation. Among the EBV-encoded proteins implicated in viralpathogenesis, considerable attention has focused upon latent membraneprotein 1 (LMP1). Of the nine EBV genes expressed as proteins inEBV-transformed cells, LMP1 is the best characterized, and is the onlyEBV-encoded gene product capable of transforming cells in vitro and invivo, resulting in the potential for lymphoproliferative changes andmalignancy. In addition to its established role in the pathogenesis of Bcell lymphoma and other malignancies, EBV infection may be linked toexacerbation of various human autoimmune diseases, including RA and SLE.

The mouse collagen-induced arthritis (CIA) model (Myers, et al., LifeScience 61: 1861-1878 (1997)) has many pathologic and immunologicparallels to rheumatoid arthritis, and provides a stable, predictablemodel for evaluating the therapeutic potential of compounds for treatingchronic inflammatory conditions. This model can be used, for example, toevaluate the ability of the honokiol analogues to treat and/or preventthese disorders.

Treatment of mouse B cell lines with the honokiol analogues in vitro canbe shown to recapitulate the cytokine profile seen in primary mouse Bcells with a concomitant dose-dependent decrease in CD40 and LMP1-mediated NFkB and AP-1 activation. Those compounds which decrease CD40and LMP1-mediated NFkB and AP-1 activation in a dose-dependent mannerwill be expected to have anti-inflammatory properties, potentially inboth the cognitive phase of the immune response, as well as the effectorphase, by inhibiting cytokines that lead to chronic inflammation andadditional pathology.

Treatment of Ocular Disorders

The compounds are also suitable for use in treating ocular disorderswith an inflammatory component, such as macular degeneration, retinalvasculitis, uveitis, conjunctivitis, episcleritis, scleritis, opticneuritis, retrobulbar neuritis, ocular inflammation following ocularsurgery, and ocular inflammation resulting from physical eye trauma. Inthis embodiment, the compounds can be delivered via eye drops or othersuitable topical formulation for direct administration to the eye.

Treatment of Neurodegenerative Disorders and/or ProvidingNeuroprotection

Reactive oxygen species also induce inflammation and neurodegeneration.Inhibition of these species can also result in neuroprotection,including protection from further damage following an ischemic braininjury such as a stroke, or that caused from blunt trauma, and treatmentor prevention of neurodegenerative disorders such as Alzheimer'sdisease, senile dementia, pre-senile dementia, Parkinsons disease,Huntington's Chorea, multiple sclerosis, and the like.

Reactive oxygen species also drive seizures, and the honokiol analogueshave GABAergic activity which may ameliorate seizures as well.

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, allparts and percentages are by weight, unless otherwise noted. Reactionyields are reported in mole percentages.

EXAMPLES

The following examples are provided to illustrate the present inventionand should not be construed as limiting the scope thereof. In theseexamples, all parts and percentages are by weight, unless otherwisenoted. Reaction yields are reported in mole percentage.

Example 1 Spectrophotometric Assay of NADH Oxidase

NADH oxidase activity can be determined as the disappearance of NADHmeasured at 340 nm in a reaction mixture containing 25 mM Tris-Mesbuffer (pH 7.2), 1 mM KCN, and 150 μM NADH at 37° C. Activity can bemeasured, for example, using a Hitachi U3210 spectrophotometer withstirring and continuous recording over two intervals of 5 min each. Amillimolar extinction coefficient of 6.22 can be used to determinespecific activity.

Example 2 Measuring Cell Growth

A mouse mammary tumor subpopulation line 4T1 arising from a BALB/cf C3Hmouse can be grown in DME-10, Dulbecco's modified Eagle's mediumsupplemented with 5% fetal calf serum, 5% newborn calf serum, 1 mM mixednon-essential amino acids, 2 mM L-glutamine, penicillin (100 units/ml),and streptomycin (100 .mu.g/ml) (Miller et al., 1987, Brit. J. Can.56:561-569 and Miller et al., 1990, Invasion Metastasis 10:101-112).

Example 3 In Vitro Testing of Various Test Compounds

Nude mice were injected subcutaneously with approximately one milliontumor cells. Once tumors became visible, they were treated with 40 mg/kgdaily of honokiol diepoxide. The compound was reconstituted in 100microliters of ethanol and diluted with 900 microliters of 20%Intralipid, and 0.3 ml of this mixture was injected intraperitoneallydaily. Tumors were measured with vernier calipers, and tumor volume wascalculated using the formula (width²×length) 0.52, where width is thesmallest dimension, 2 represents squared, and 1 represents the length.

The results are shown in Table 1, below, and in FIG. 1.

TABLE 1 Tumor Volume in Honokiol Diepoxide-Treated Animals Group L WTumor Volume Average Control Honokiol Epoxide Control 12.42 10.18669.2994922 21.82 21.24 5118.787665 22.58 11.98 1685.159129 2491.0822491.082 433.3632 Honokiol 0 0 0 Epoxide 10.04 8.59 385.2329125 14.54 11914.8568 433.3632

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention claimed is:
 1. A method for treating a cancer or a benigntumor in a mammal, comprising administering to the mammal atherapeutically effective amount of the formula:

W is (CHR²)n, O—(CHR²)n, S—(CHR²)n, or NR²—(CHR²)n, wherein the (CHR²)nportion of the W moiety can be fluorinated with anywhere from onefluorine atom to complete perflourination, X is O, CR², or a bondbetween the two carbons to which it is attached, R¹ is H, C₁₋₈ alkylphosphate, or dichloroacetate, R² is H, C₁₋₈ alkyl, or alkylaryl, or,when bonded to carbon, can be halo, n is an integer from 1-4, G isselected from the group consisting of C₁₋₆ alkyl, C₂₋₈ alkenyl, halo,—OR¹, —NR³R⁴, —CF₃, —CN, —NO₂, —C₂R³, —SR³, —N₃, —C(═O)NR³R⁴,—NR³C(═O)R³, —C(═O)R³, —C(═O)OR³, —OC(═O)R³, —OC(═O)NR³R⁴, —NR³C(═O)OR³,—SO₂R³, —SO₂NR³R⁴, and —NR³SO₂R³, where R³ and R⁴ are individuallyhydrogen, C₁₋₆ alkyl, or C₃₋₈ cycloalkyl, z is an integer of from 0-3,wherein only 0 or 1 of the X variables can be a bond between the twocarbons to which it is attached, in an amount sufficient to induceapoptosis and/or inhibit angiogenesis, such that the growth of the tumoris at least partially inhibited, wherein the cancer is selected from thegroup consisting of melanoma, rectal carcinoma, colon carcinoma, breastcarcinoma, ovarian carcinoma, small cell lung carcinoma, chroniclymphocytic carcinoma, esophageal carcinoma, prostate carcinoma, breastcancer, myeloma, and lymphoma, and the benign tumor is a hemangioma. 2.A method of claim 1, wherein one or two of R¹ represent an C₁₋₈ alkylphosphate ester, or a dichloroacetate.
 3. A method of claim 1, whereinboth of X are O.
 4. A method of claim 1, wherein one of X is O, and theother represents a bond between the two carbons to which it is attached.5. A method of claim 1, wherein each R² is H.
 6. A method of claim 1,wherein each W is CH₂.
 7. A method for treating a cancer or a benigntumor in a mammal, comprising administering to the mammal atherapeutically effective amount of a compound of the formula:

wherein W is (CHR²)n, O—(CHR²)n, S—(CHR²)n, or NR²—(CHR²)n, wherein the(CHR²)n portion of the W moiety can be fluorinated with anywhere fromone fluorine atom to complete perflourination, R¹ is H, C₁₋₈ alkylphosphate, or dichloroacetate, R² is H, C₁₋₈ alkyl, or alkylaryl, n isan integer from 1-4, G is selected from the group consisting of C₁₋₆alkyl, C₂₋₈ alkenyl, halo, —OR′, —NR³R⁴, —CF₃, —CN, —NO₂, —C₂R³, —SR³,—N₃, —C(═O)NR³R⁴, —NR³C(═O)R³, —C(═O)R³, —C(═O)OR³, —OC(═O)R³,—OC(═O)NR³R⁴, —NR³C(═O)OR³, —SO₂R³, —SO₂NR³R⁴, and —NR³SO₂R³, where R³and R⁴ are individually hydrogen, C₁₋₆ alkyl, or C₃₋₈ cycloalkyl, z isan integer of from 0-3, and in an amount sufficient to induce apoptosisand/or inhibit angiogenesis, such that the growth of the tumor is atleast partially inhibited, wherein the cancer is selected from the groupconsisting of melanoma, rectal carcinoma, colon carcinoma, breastcarcinoma, ovarian carcinoma, small cell lung carcinoma, chroniclymphocytic carcinoma, esophageal carcinoma, prostate carcinoma, breastcancer, myeloma, and lymphoma, and the benign tumor is a hemangioma. 8.A method of claim 7, wherein each R² is H.
 9. A method of claim 7,wherein each W is CH₂.
 10. A method for treating a cancer in a mammal,comprising administering to the mammal a therapeutically effectiveamount of a compound selected from the group consisting of:

wherein the —(CH₂)— portion of the

moiety can be fluorinated with anywhere from one fluorine atom tocomplete perflourination, in an amount sufficient to induce apoptosisand/or inhibit angiogenesis, such that the growth of the tumor is atleast partially inhibited, wherein the cancer is selected from the groupconsisting of hemangioma, melanoma, rectal carcinoma, colon carcinoma,breast carcinoma, ovarian carcinoma, small cell lung carcinoma, chroniclymphocytic carcinoma, hairy cell leukemia, osophogeal carcinoma,prostate carcinoma, breast cancer, myeloma, and lymphoma.
 11. The methodof claim 10, wherein the compound is honokiol diepoxide.
 12. A methodfor treating a cancer in a mammal, comprising administering to themammal a therapeutically effective amount of a compound selected fromthe group consisting of valproate mono and diesters of honokiol,dichloroacetate mono and diesters of honokiol, and C₁₋₆ alkyl phosphatemono and di-esters of honokiol, in an amount sufficient to induceapoptosis and/or inhibit angiogenesis, such that the growth of the tumoris at least partially inhibited, wherein the cancer is selected from thegroup consisting of hemangioma, melanoma, rectal carcinoma, coloncarcinoma, breast carcinoma, ovarian carcinoma, small cell lungcarcinoma, chronic lymphocytic carcinoma, hairy cell leukemia,osophogeal carcinoma, prostate carcinoma, breast cancer, myeloma, andlymphoma.
 13. The method of claim 1 wherein the mammal is a human. 14.The method of claim 1, wherein the cancer is a metastasized cancer. 15.The method of claim 1 wherein the human is immunosuppressed by reason ofhaving undergone anti-cancer therapy prior to administration of thecomposition.
 16. The method of claim 15, wherein the tumor is a tumor ofepithelial tissue, lymphoid tissue, connective tissue, bone, or thecentral nervous system.
 17. The method of claim 1, wherein the compoundsare administered parenterally, orally, or directly into the tumor. 18.The method of claim 1, wherein the compounds are administered via animplanted device.
 19. The method of claim 1, wherein the administrationis provided using a sustained release formulation.
 20. The method ofclaim 1, wherein the cancer is breast cancer.
 21. The method of claim 7,wherein the cancer is breast cancer.